Method for predicting the risk of obesity in a subject

ABSTRACT

Subject matter of the present invention is a method for determining the fat processing activity and/or predicting the risk of obesity in a subject comprising the following steps determining the level of pro-neurotensin or fragments thereof of at least 5 amino acids in a bodily fluid obtained from said subject; and correlating said level of pro-neurotensin or fragments thereof with fat processing activity and/or the risk of incidence of obesity in said subject, wherein an elevated level is indicative of enhanced fat processing activity and/or predictive for an enhanced risk of getting obesity.

Subject matter of the present invention is a method for determining thefat processing activity and/or for predicting the risk of obesity in asubject comprising the following steps:

-   -   determining by an immunoassay the level of pro-neurotensin 1-117        or fragments thereof of at least 5 amino acids or        pro-neurotensin 1-117 comprising peptides in a bodily fluid        obtained from said subject or    -   determining the level of pro-neurotensin 1-117 or fragments        thereof of at least 5 amino acid in a bodily fluid obtained from        said subject; and    -   correlating said level of pro-neurotensin 1-117 or fragments        thereof or pro-neurotensin 1-117 comprising peptides with the        fat processing activity and/or the a risk of incidence of        obesity in said subject, wherein an elevated level is indicative        of an enhanced fat processing activity and/or predictive for an        enhanced risk of obesity    -   and wherein subject is not obese at the time the sample of        bodily fluid is taken from said subject.

Neurotensin is a 13-amino acid neuropeptide derived from theprepro-neurotensin precursor and stochiometrically released togetherwith the stable 117-amino acid peptide pro-neurotensin (P-NT) and themature hormone binds to three different receptors, neurotensin receptor1 and 2 (Ntsr1 and Ntsr2), which are G-protein coupled receptors andneurotensin receptor 3 (Ntsr3) which is non-G-protein coupled and alsoknown as Sortillin-1 (SORT1).

Neurotensin is released peripherally from the small intestine as well ascentrally from the hypothalamus. The peripheral secretion of neurotensinis stimulated by food-intake, especially by fat, and is known toregulate gastrointestinal motility and pancreatic and biliary secretion.Interestingly, neurotensin is implicated in appetite control as ananorectic hormone as it acutely reduces food intake following bothcentral (intracerebroventricular) and peripheral (intraperitoneal)injection in rats, an effect which seems mainly mediated through theneurotensin-1 receptor (Ntsr1). In obese as compared to normal-weighthuman subjects, postprandial plasma neurotensin concentration wasreduced following a liquid fatty meal (Wisen et al. 1992, Reg Peptides39(1): 43-54) and lower fasting neurotensin levels have been observed inobese human subjects compared with lean controls (Weiss et al. 2001,Obes Surg 11: 735-739), suggesting that the regulation of neurotensinsecretion is disturbed in obesity. However, no large study hasinvestigated if and how neurotensin is related to the risk of developingobesity.

A subject of the present invention was to investigate the prognosticpower of NT for predicting the risk of obesity in a non-obese subject.To address this issue, we measured stable fragments of pro-neurotensinin fasting plasma in said Swedish prospective cohort study (Malmö Dietand Cancer Study) and related baseline level of this biomarker toobesity during 15 years of follow-up.

The risk of obesity in a non-obese subject may correlate to the fatprocessing activity of said subject. Thus, the determination of levelsof pro-neurotensin 1-117 or fragments thereof of at least 5 amino acidsor pro-neurotensin 1-117 comprising peptides in a bodily fluid may be adetermination of the fat processing activity.

Subject matter of the present invention is a method for determining thefat processing activity and/or for predicting the risk of obesity in asubject comprising the following steps:

-   -   determining by an immunoassay the level of pro-neurotensin 1-117        or fragments thereof of at least 5 amino acids or        pro-neurotensin 1-117 comprising peptides in a bodily fluid        obtained from said subject or    -   determining the level of pro-neurotensin 1-117 or fragments        thereof of at least 5 amino acid in a bodily fluid obtained from        said subject; and    -   correlating said level of pro-neurotensin 1-117 or fragments        thereof or pro-neurotensin 1-117 comprising peptides with the        fat processing activity and/or the a risk of incidence of        obesity in said subject, wherein an elevated level is indicative        of an enhanced fat processing activity and/or predictive for an        enhanced risk of obesity    -   and wherein subject is not obese at the time the sample of        bodily fluid is taken from said subject.

Fat processing activity is defined as absorption, metabolism and/orstorage of fat in the human body. Fat processing activity is synonymousto fat up-take by the body or to the capability to storage fat that isup-taken and converted. Woman with higher fat processing activity are athigher risk of getting obese.

Dietary fat consists of a variety of polar and nonpolar lipids.Triacylglycerol (TAG) is the dominant fat in the diet, contributing90-95% of the total energy derived from dietary fat. Dietary fats alsoinclude phospholipids, sterols (e.g., cholesterol), and many otherlipids (e.g., fat-soluble vitamins) The digestion of lipids begins inthe oral cavity through exposure to lingual lipases, which are secretedby glands in the tongue to begin the process of digesting triglycerides.Digestion continues in the stomach through the effects of both lingualand gastric enzymes. The stomach is also the major site for theemulsification of dietary fat and fat-soluble vitamins, with peristalsisa major contributing factor. Crude emulsions of lipids enter theduodenum as fine lipid droplets and then mix with bile and pancreaticjuice to undergo marked changes in chemical and physical form.Emulsification continues in the duodenum along with hydrolysis andmicellization in preparation for absorption across the intestinal wall.Bile and pancreatic juice provide pancreatic lipase, bile salts, andcolipase, which function cooperatively to ensure the efficiency of lipiddigestion and absorption. TAG is digested primarily by pancreatic lipasein the upper segment of the jejunum. Free fatty acids are taken up fromthe intestinal lumen into the enterocytes and used for the biosynthesisof neutral fats. Once inside the enterocyte, the products of TAGhydrolysis must traverse the cytoplasm to reach the endoplasmicreticulum (ER), where they are used to synthesize complex lipids.

The major lipoproteins secreted by the intestine are VLDL andchylomicrons. Of these, the chylomicrons are synthesized exclusively inthe intestine to transport dietary fat and fat-soluble vitamins into theblood. Chylomicrons are primarily very large, spherical TAG-richparticles that also contain PLs, cholesterol, vitamin E, vitamin A, andprotein. The lipoprotein core contains TAG, cholesteryl esters, andfat-soluble vitamins, whereas the surface contains a monolayer ofphospholipids (mainly phosphatidylcholine), free cholesterol, andprotein.

As they circulate, the triacylglycerols of chylomicrons undergohydrolysis by lipoprotein lipase, an enzyme located on the surface ofcapillary endothelial cells of muscle and adipose tissues. Circulatingchylomicrons have a half-life of 5-10 minutes. The hydrolysis ofchylomicrons leads to release of fatty acids and glycerol from the coreof chylomicrons, as well as unesterified cholesterol from the surfacecoat of these particles. The delipidation occurs predominantly in theadipose, muscle and heart tissues which take up and oxidize or store thefatty acids released by lipoprotein lipase.

Fatty acids, cholesterol and bile acids that escape intestinalabsorption are excreted as fecal fatty acids, as well as neutral andacidic sterols, respectively.

In subjects with an effective fat processing activity the proportion offat absorbed by the gastrointestinal tract is lower, the amount of fatabsorbed is metabolized nearly completely and no or only a small amountof fat is transported and stored in the adipocytes of the subjects. Incontrast, in subjects with an inefficient fat processing activity theproportion of fat absorption by the GI tract is high, but the fatabsorbed is metabolized to a smaller extent and more fat is transportedand stored in the adipocytes of the subject.

The term “subject” as used herein refers to a living human or non-humanorganism. Preferably herein the subject is a human subject. In aspecific embodiment of the invention said subject is a non-diabeticsubject. In another specific embodiment of the invention said subject isnon-IFG (non-pre-diabetic) subject. In another specific embodiment ofthe invention the subject does not suffer from the metabolic syndrome.In another specific embodiment of the invention the subject does notsuffer from a cardiac disease. In another specific embodiment of theinvention the subject does not suffer from cancer. In another specificembodiment of the invention the subject is a female subject.

The term “cardiac disease” includes myocardial infarction, ischemicheart disease, stroke, heart failure (acute or chronic heart failure),atrial fibrillation and atrial flutter.

The term “risk of new-onset obesity” is synonymously to “risk ofobesity” wherein subject is not obese at the time the sample of bodilyfluid is taken from said subject.

In one embodiment of the invention the level of pro-neurotensin orfragments thereof of at least 5 amino acids or pro-neurotensin 1-117comprising peptides in a bodily fluid is the fasting level ofpro-neurotensin or fragments thereof of at least 5 amino acid orpro-neurotensin 1-117 comprising peptides. Fasting level means no fooduptake 10 hours or preferably 12 hours prior blood sampling.

As the fasting level of pro-neurotensin or fragments thereof of at least5 amino acid or pro-neurotensin 1-117 comprising peptides is nottriggered by recent fat up-take it seems that in one embodiment thefasting level is a measure to quantify the fat processing activity ofsaid subject. This may be a missing link that explains why some subjectsare susceptible to become obese as they process high quantities ofup-taken fat whereas other individuals with low levels ofpro-neurotensin or fragments thereof of at least 5 amino acid orpro-neurotensin 1-117 comprising peptides have a low level of fatprocessing activity. But also determination of pro-neurotensin orfragments thereof of at least 5 amino acid or pro-neurotensin 1-117comprising peptides in non-fasting sample may be an indicator offat-processing activity as some subjects may have higher enhancements oflevels than others triggered by the same amount of fat up taken. In thelatter setting pro-neurotensin or fragments thereof of at least 5 aminoacid or pro-neurotensin 1-117 comprising peptides may be measured atbaseline before fat-uptake and thereafter, whereas subjects withenhanced fat processing activity will react with higher amounts ofreleased pro-neurotensin or fragments thereof of at least 5 amino acidor pro-neurotensin 1-117 comprising peptides upon fat-uptake.

Thus, one embodiment of the invention is a method for determining thefat processing activity and/or for predicting the risk of obesity in asubject comprising the following steps:

-   -   determining by an immunoassay the level of pro-neurotensin 1-117        or fragments thereof of at least 5 amino acids or        pro-neurotensin 1-117 comprising peptides in a non-fasting or        fasting sample of bodily fluid obtained from said subject or    -   determining the level of pro-neurotensin 1-117 or fragments        thereof of at least 5 amino acid in non-fasting or fasting        sample of a bodily fluid obtained from said subject; and    -   administering to said subject fat    -   determining by an immunoassay the level of pro-neurotensin 1-117        or fragments thereof of at least 5 amino acids or        pro-neurotensin 1-117 comprising peptides in a sample of bodily        fluid obtained from said subject after fat-uptake or    -   determining the level of pro-neurotensin 1-117 or fragments        thereof of at least 5 amino acid in of a bodily fluid obtained        from said subject after fat-uptake; and    -   calculating the difference between said levels after and before        fat-uptake and    -   correlating said difference between said levels after and before        fat-uptake of pro-neurotensin 1-117 or fragments thereof or        pro-neurotensin 1-117 comprising peptides with the fat        processing activity and/or the a risk of incidence of obesity in        said subject, wherein a higher difference is indicative of an        enhanced fat processing activity and/or predictive for an        enhanced risk of obesity    -   and wherein the subject is not obese at the time the sample of        bodily fluid is taken from said subject.

This means that the difference between the level of pro-neurotensin1-117 or fragments thereof of at least 5 amino acids or pro-neurotensin1-117 comprising peptides before fat up-take and said level after fatup-take may be indicative of the fat processing activity. The status ofsaid subject before fat up-take may be fasting or non-fasting. Astandardized amount of fat is administered to said subjects (oral fattolerance test), e.g. a specific amount of cream containing a specificamount of fat. It has been shown in Example 4 that some individualsreact more sensitive to fat-uptake than others and thus, have a higherfat processing activity.

The level of pro-neurotensin 1-117 or fragments thereof of at least 5amino acids or pro-neurotensin 1-117 comprising peptides in a bodilyfluid obtained from said subject that is predictive for the risk ofdeveloping new-onset obesity and is indicative for fat processingactivity is released from the small intestine. The release ofneurotensin from the small intestine is stimulated by food intake,especially by fat (Go and Demol 1981. Peptides (Suppl. 2): 267-269), andis known to regulate gastrointestinal motility and pancreatic andbiliary secretion (Reinecke 1985. Frog Histochem Cytochem 16: 1-172).Pro-neurotensin 1-117 and fragments thereof or pro-neurotensin 1-117comprising peptides are used as a surrogate marker for the releasedneurotensin as neurotensin and pro-neurotensin 1-117 and fragmentsthereof or pro-neurotensin 1-117 comprising peptides are released inequimolar amounts from pro-neurotensin.

It is the surprising finding of the present invention that theperipheral secretion of neurotensin/pro-neurotensin 1-117 or fragmentsthereof of at least 5 amino acids or pro-neurotensin 1-117 comprisingpeptides is indicative for the susceptibility of a subject fordeveloping new-onset obesity and is a measure of fat processingactivity. Thus, dietary measures as reduction of fat uptake may lowersaid risk in said subject.

The correlation between the level of pro-neurotensin or fragmentsthereof of at least 5 amino acids or PNT 1-117 comprising peptides in abodily fluid obtained from said subject and the risk of developingobesity and also the fat processing activity is continuous, i.e. thehigher the level the higher the risk.

For the sake of practicability the person skilled in the art may usethreshold(s).

The term “elevated level” means a level above a certain threshold level.

Threshold levels may be determined by measuring samples from subjectswho did develop a certain condition (e.g. obesity) and samples fromsubjects who did not develop the condition. One possibility to determinea threshold is the calculation of receiver operating characteristiccurves (ROC curves), plotting the value of a variable versus itsrelative frequency in the “normal” population (e.g. subjects who did notdevelop the condition of obesity) and “disease” population (e.g.subjects who did develop the condition of obesity). A distribution ofthe marker levels for subjects developing or not developing a certaincondition will likely overlap. Under such conditions, a test does notabsolutely distinguish “normal” from “disease” with 100% accuracy, andthe area of overlap indicates where the test cannot distinguish normalfrom “disease”. A threshold is selected, above which (or below which,depending on how a marker changes with the “disease”) the test isconsidered to be abnormal and below which the test is considered to benormal. The area under the ROC curve is a measure of the probabilitythat the perceived measurement will allow correct identification of acondition. ROC curves can be used even when test results don'tnecessarily give an accurate number. As long as one can rank results,one can create a ROC curve. For example, results of a test on “disease”samples might be ranked according to degree (e.g. 1=low, 2=normal, and3=high). This ranking can be correlated to results in the “normal”population, and a ROC curve created. These methods are well known in theart (Hanley et al. 1982. Radiology 143: 29-36). Preferably, a thresholdis selected to provide a ROC curve area of greater than about 0.5, morepreferably greater than about 0.7, still more preferably greater thanabout 0.8, even more preferably greater than about 0.85, and mostpreferably greater than about 0.9. The term “about” in this contextrefers to +/−5% of a given measurement. The horizontal axis of the ROCcurve represents (1-specificity), which increases with the rate of falsepositives. The vertical axis of the curve represents sensitivity, whichincreases with the rate of true positives. Thus, for a particularcut-off selected, the value of (1-specificity) may be determined, and acorresponding sensitivity may be obtained. The area under the ROC curveis a measure of the probability that the measured marker level willallow correct identification of a disease or condition. Thus, the areaunder the ROC curve can be used to determine the effectiveness of thetest. The odds ratio is a measure of effect size, describing thestrength of association or non-independence between two binary datavalues (e.g. the ratio of the odds of an event occurring in testnegative group to the odds of it occurring in the test positive group).

Threshold levels can be obtained for instance from a Kaplan-Meieranalysis, where the occurrence of a disease or the probability of aserious condition and/or death is correlated with the e.g. quartiles ofthe respective markers in the population. According to this analysis,subjects with marker levels above the 75th percentile have asignificantly increased risk for getting the diseases according to theinvention. This result is further supported by Cox regression analysiswith adjustment for classical risk factors. The highest quartile versusall other subjects is highly significantly associated with increasedrisk for getting a disease or the probability of a serious conditionand/or death according to the invention.

Other preferred cut-off values are for instance the 90th, 95th or 99thpercentile of a reference population. By using a higher percentile thanthe 75th percentile, one reduces the number of false positive subjectsidentified, but one might miss to identify subjects, who are atmoderate, albeit still increased risk. Thus, one might adapt the cut-offvalue depending on whether it is considered more appropriate to identifymost of the subjects at risk at the expense of also identifying “falsepositives”, or whether it is considered more appropriate to identifymainly the subjects at high risk at the expense of missing severalsubjects at moderate risk.

Other mathematical possibilities to calculate an individual's risk byusing the individual's marker level value and other prognosticlaboratory and clinical parameters are for instance the NRI (NetReclassification Index) or the IDI (Integrated Discrimination Index).The indices can be calculated according to Pencina (Pencina M J, et al.:Evaluating the added predictive ability of a new marker: from area underthe ROC curve to reclassification and beyond. Stat Med. 2008;27:157-172).

A bodily fluid may be selected from the group comprising blood, serum,plasma, urine, cerebrospinal fluid (CSF), and saliva. In one specificembodiment a bodily fluid may be selected from the group comprisingblood, serum, plasma.

The present data suggest a strong correlation between the level ofpro-neurotensin or fragments thereof, especially pro-neurotensin 1-117or fragments thereof or pro-neurotensin 1-117 comprising peptides withthe development of new-onset obesity and with the fat processingactivity.

Fragments of pro-neurotensin that may be determined in a bodily fluidmay be e.g.

SEQ ID NO: 1 (Pro-neurotensin 1-147)SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCSLVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLTIYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVIKRKIPYILKRQLY ENKPRRPYIL KRDSYYY SEQ ID NO: 2 (pro-neurotensin 1-125 (largeneuromedin N)) SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCSLVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLTIYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVI KR KIPYILSEQ ID NO: 3 (neuromedin N:) KIPYIL SEQ ID NO: 4 (neurotensin)pyroQLYENKPRRP YIL SEQ ID NO: 5 (pro-neurotensin 1-117)SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCSLVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLTIYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVISEQ ID NO: 6 (pro-neurotensin 1-132)SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCSLVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLTIYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVIKRK IPYILKRQLY ENSEQ ID No 7: (Pro-Neurotensin 1-140 (large neurotensin)SDSEEEMKAL EADFLTNMHT SKISKAHVPS WKMTLLNVCSLVNNLNSPAE ETGEVHEEEL VARRKLPTAL DGFSLEAMLTIYQLHKICHS RAFQHWELIQ EDILDTGNDK NGKEEVIKRK IPYILKRQLY ENKPRRPYILSEQ ID NO: 8 (pro-neurotensin 120-140) KIPYILKRQL YENKPRRPYI LSEQ ID NO: 9 (pro-neurotensin 120-147) KIPYILKRQL YENKPRRPYIL KRDSYYYSEQ ID NO: 10 (pro-neurotensin 128-147) QLYENKPRRP YILKRDSYYY

In a more specific embodiment of the method according to the presentinvention the level of pro-neurotensin 1-117 is determined.

In a specific embodiment the level of pro-neurotensin, especiallypro-neurotensin 1-117 or fragments thereof or pro-neurotensin 1-117comprising peptides, is measured with an immunoassay. More specificallyan immunoassay is used as described in Ernst et al. Peptides 27 (2006)1787-1793. In one specific embodiment the immune reactivity ofpro-neurotensin 1-117 is determined wherein immune reactivity means thefollowing:

Determining the level of pro-neurotensin or fragments thereof may meanthat the immunoreactivity towards pro-neurotensin or fragments thereofincluding neurotensin and neuromedin N is determined. A binder used fordetermination of pro-neurotensin or fragments thereof includingneurotensin and neuromedin N depending of the region of binding may bindto more than one of the above displayed molecules. This is clear to aperson skilled in the art.

Thus, according to the present invention the level of immunoreactiveanalyte by using at least one binder that binds to a region within theamino acid sequence of any of the above peptide and peptide fragments,(i.e. pro-neurotensin (pro-NT) and fragments according to any of thesequences 1 to 10), is determined in a bodily fluid obtained from saidsubject; and correlated to the specific embodiments of clinicalrelevance.

In a more specific embodiment of the method according to the presentinvention the level of pro-NT 1-117 is determined (SEQ ID NO. 5:pro-neurotensin 1-117). In a more specific embodiment the level ofimmunoreactive analyte by using at least one binder that binds to pro-NT1-117 is determined and is correlated to the specific embodiments ofclinical relevance according to the invention.

In another embodiment of the invention the immunoreactivity towardspro-neurotensin or fragments thereof not including neurotensin andneuromedin N is determined.

An immunoassay that may be useful for determining the level ofpro-neurotensin or fragments thereof of at least 5 amino acids orpro-neurotensin 1-117 comprising peptide may comprise the steps atoutlines in example 2. An immunoassay that may be useful for determiningthe level of pro-neurotensin or fragments thereof of at least 5 aminoacids or pro-neurotensin 1-117 comprising peptide may comprise at leastone antibody or at least two antibodies directed against an epitopewithin pro-neurotensin 1-117. At least of these antibodies may belabelled. All thresholds and values have to be seen in light of the testand the calibration used according to Example 2. A person skilled in theart may know that the absolute value of a threshold might be influencedby the calibration used. This means that all values and thresholds givenherein are to be understood in context of the calibration used in herein(Example 2). A human pro-NT-calibrator is available by ICI-Diagnostics,Berlin, Germany. Alternatively, the assay may be calibrated by syntheticor recombinant pro-NT 1-117 or fragments thereof (see also Ernst et. al,2006).

In one embodiment of the invention it may be a so-called POC-test(point-of-care), that is a test technology which allows performing thetest within less than 1 hour near the patient without the requirement ofa fully automated assay system. One example for this technology is theimmunochromatographic test technology.

In one embodiment of the invention such an assay is a sandwichimmunoassay using any kind of detection technology including but notrestricted to enzyme label, chemiluminescence label,electrochemiluminescence label, preferably a fully automated assay. Inone embodiment of the invention such an assay is an enzyme labeledsandwich assay. Examples of automated or fully automated assay compriseassays that may be used for one of the following systems: RocheElecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®,Biomerieux Vidas®, Alere Triage®.

A variety of immunoassays are known and may be used for the assays andmethods of the present invention, these include: radioimmunoassays(“RIA”), homogeneous enzyme-multiplied immunoassays (“EMIT”), enzymelinked immunoadsorbent assays (“ELISA”), apoenzyme reactivationimmunoassay (“ARIS”), dipstick immunoassays and immuno-chromotographyassays.

In one embodiment of the invention at least one of said two binders islabeled in order to be detected.

The preferred detection methods comprise immunoassays in various formatssuch as for instance radioimmunoassay (RIA), chemiluminescence- andfluorescence-immunoassays, Enzyme-linked immunoassays (ELISA),Luminex-based bead arrays, protein microarray assays, and rapid testformats such as for instance immunochromatographic strip tests.

In a preferred embodiment said label is selected from the groupcomprising chemiluminescent label, enzyme label, fluorescence label,radioiodine label.

The assays can be homogenous or heterogeneous assays, competitive andnon-competitive assays. In one embodiment, the assay is in the form of asandwich assay, which is a non-competitive immunoassay, wherein themolecule to be detected and/or quantified is bound to a first antibodyand to a second antibody. The first antibody may be bound to a solidphase, e.g. a bead, a surface of a well or other container, a chip or astrip, and the second antibody is an antibody which is labeled, e.g.with a dye, with a radioisotope, or a reactive or catalytically activemoiety. The amount of labeled antibody bound to the analyte is thenmeasured by an appropriate method. The general composition andprocedures involved with “sandwich assays” are well-established andknown to the skilled person.

In another embodiment the assay comprises two capture molecules,preferably antibodies which are both present as dispersions in a liquidreaction mixture, wherein a first labelling component is attached to thefirst capture molecule, wherein said first labelling component is partof a labelling system based on fluorescence- orchemiluminescence-quenching or amplification, and a second labellingcomponent of said marking system is attached to the second capturemolecule, so that upon binding of both capture molecules to the analytea measurable signal is generated that allows for the detection of theformed sandwich complexes in the solution comprising the sample.

In another embodiment, said labeling system comprises rare earthcryptates or rare earth chelates in combination with fluorescence dye orchemiluminescence dye, in particular a dye of the cyanine type.

In the context of the present invention, fluorescence based assayscomprise the use of dyes, which may for instance be selected from thegroup comprising FAM (5- or 6-carboxyfluorescein), VIC, NED,Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700/800, Cyaninedyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen,6-Carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET,6-Carboxy-4′,5′-dichloro-2′,7′-dimethodyfluorescein (JOE),N,N,N′,N′-Tetramethyl-6-carboxyrhodamine (TAMRA), 6-Carboxy-X-rhodamine(ROX), 5-Carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6),Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes,such as BODIPY TMR, Oregon Green, Coumarines such as Umbelliferone,Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red,Yakima Yellow, Alexa Fluor, PET, Ethidiumbromide, Acridinium dyes,Carbazol dyes, Phenoxazine dyes, Porphyrin dyes, Polymethin dyes, andthe like.

In the context of the present invention, chemiluminescence based assayscomprise the use of dyes, based on the physical principles described forchemiluminescent materials in (24). Preferred chemiluminescent dyes areacridiniumesters.

As mentioned herein, an “assay” or “diagnostic assay” can be of any typeapplied in the field of diagnostics. Such an assay may be based on thebinding of an analyte to be detected to one or more capture probes witha certain affinity. Concerning the interaction between capture moleculesand target molecules or molecules of interest, the affinity constant ispreferably greater than 10⁸ M⁻¹.

In the context of the present invention, “binder molecules” aremolecules which may be used to bind target molecules or molecules ofinterest, i.e. analytes (i.e. in the context of the present inventionpro-Neurotensin and fragments thereof), from a sample. Binder moleculesmust thus be shaped adequately, both spatially and in terms of surfacefeatures, such as surface charge, hydrophobicity, hydrophilicity,presence or absence of lewis donors and/or acceptors, to specificallybind the target molecules or molecules of interest. Hereby, the bindingmay for instance be mediated by ionic, van-der-Waals, pi-pi, sigma-pi,hydrophobic or hydrogen bond interactions or a combination of two ormore of the aforementioned interactions between the capture moleculesand the target molecules or molecules of interest. In the context of thepresent invention, binder molecules may for instance be selected fromthe group comprising a nucleic acid molecule, a carbohydrate molecule, aPNA molecule, a protein, an antibody, a peptide or a glycoprotein.Preferably, the binder molecules are antibodies, including fragmentsthereof with sufficient affinity to a target or molecule of interest,and including recombinant antibodies or recombinant antibody fragments,as well as chemically and/or biochemically modified derivatives of saidantibodies or fragments derived from the variant chain with a length ofat least 12 amino acids thereof.

Chemiluminescent label may be acridinium ester label, steroid labelsinvolving isoluminol labels and the like.

Enzyme labels may be lactate dehydrogenase (LDH), creatinekinase (CPK),alkaline phosphatase, aspartate aminotransferace (AST), alanineaminotransferace (ALT), acid phosphatase, glucose-6-phosphatedehydrogenase and so on.

In one embodiment of the invention at least one of said two binders isbound to a solid phase as magnetic particles, and polystyrene surfaces.

The threshold for determining the risk of a subject for developingnew-onset obesity is above 60 pmol/L pro-NT, preferred above 90 pmol/L,more preferred above 123 pmol/L. In a specific embodiment said thresholdis above 180 pmol/L or above 190 pmol/L. These thresholds are related tothe above mentioned calibration method. A pro-NT value above saidthreshold means that the subject has an enhanced risk of developingnew-onset obesity.

Obesity is defined as a body mass index of ≥30 kg/m².

A non-obese subject is defined with a body mass index of <30 kg/m².

New-onset obesity is defined as obesity development of non-obesesubjects after a certain follow-up time.

Body mass index (BMI) was defined as body weight in kilograms divided bythe square of height in meters.

The time for follow-up is up to 1 year, preferably up to 2 years, morepreferably up to 5 years, even more preferred more than 10 years, evenmore preferred up to 15 years, even more preferred up to 16.5 years,most preferred up to 18 years.

The definition of diabetes is as follows: a history of physiciandiagnosis or being on anti-diabetic medication or having a fasting wholeblood glucose >/=6.1 mmol/l (which corresponds to >/=7.0 mmol/l inplasma) at the baseline examination.

Pre-diabetes or impaired fasting glucose (IFG) is defined as whole bloodfasting plasma glucose between >/=5.4 and <6.1 mmol/l (which correspondsto 6.1-6.9 mmol/l in plasma).

In a specific embodiment of the method according to the invention saidsubject is a non-diabetic subject with fasting whole blood glucose ofless than 5.4 mmol/l (which corresponds to <6.1 mmol/l in plasma). Themetabolic syndrome was defined by the World Health Organization criteria(Alberti and Zimmet 1998. Diabet Med. 15:539-553; World HealthOrganization. 1999. Definition, diagnosis and classification of diabetesmellitus and its complications: report of a WHO Consultation. Part 1:diagnosis and classification of diabetes mellitus. Geneva, Switzerland:World Health Organization) that require the presence of insulinresistance identified by one of the following: (1) type II diabetes; (2)impaired fasting glucose; (3) impaired glucose tolerance or (4) forthose with normal fasting glucose levels (<110 mg/dL), glucose uptakebelow the lowest quartile for background population under investigationunder hyperinsulemic, euglycemic conditions, AND two of the following:(1) blood pressure: ≥140/90 mmHg; (2) dyslipidemia: triglycerides (TG):≥1.695 mmol/L and high-density lipoprotein cholesterol (HDL-C)≤0.9mmol/L (male), ≤1.0 mmol/L (female); (3) central obesity: waist:hipratio ≥0.90 (male); ≥0.85 (female), or body mass index >30 kg/m²; (4)microalbuminuria: urinary albumin excretion ratio ≥20 μg/min oralbumin:creatinine ratio ≥30 mg/g.

The subject may have normal blood pressure (BP normal blood pressure(BP). Said subject may be normotensive/high blood pressure.

The definition of normotensive/high blood pressure (HBP) is as follows:

HBP: Systolic BP>/=140 mmHg or Diastolic BP>/=90 mmHg or being onantihypertensive medications. Subjects having normal blood pressure (BP)are all other subjects, i.e subjects with systolic BP<140 mmHg orDiastolic BP<90 mmHg or not being on antihypertensive medications.

In a specific embodiment of the method according to the invention theprediction of the risk of the subject for developing new-onset obesityis improved by additionally determining and using the level of at leastone laboratory parameter or further marker selected from the groupcomprising fasting whole blood or plasma glucose, triglycerides, HDLcholesterol or subfractions thereof, LDL cholesterol or subfractionsthereof, cystatin C, insulin, CRP, estimated glomerular filtration rate(eGFR).

In a specific embodiment of the method according to the inventionadditionally at least one clinical parameter is determined selected fromthe group comprising age, gender, systolic blood pressure, diastolicblood pressure, antihypertensive treatment (AHT), waist circumference,waist-hip-ratio, current smoker, diabetes heredity, cancer heredity andprevious cardiovascular disease (CVD).

A further embodiment of the invention is a method for determining thefat processing activity and/or predicting the risk of new-onset obesity,wherein the level of pro-neurotensin or fragments thereof either aloneor in conjunction with other prognostically useful laboratory orclinical parameters is used for the determination of fat processingactivity and/or the prediction of the risk of new-onset obesity in asubject by a method which may be selected from the followingalternatives:

-   -   comparison with the median of the level of pro-neurotensin or        fragments thereof or pro-neurotensin 1-117 comprising peptides        in an ensemble of pre-determined samples in a population of        apparently healthy subjects,    -   comparison with a quantile of the level of pro-neurotensin or        fragments thereof or pro-neurotensin 1-117 comprising peptides        in an ensemble of pre-determined samples in a population of        apparently healthy subjects,    -   calculation based on Cox Proportional Hazards analysis or by        using Risk index calculations such as the NRI (Net        Reclassification Index) or the IDI (Integrated Discrimination        Index).

In one embodiment of the invention the sample is selected from the groupcomprising blood sample, a serum sample, a plasma sample, acerebrospinal fluid sample, a saliva sample and a urine sample or anextract of any of the aforementioned samples. In a specific embodimentof the method according to the invention the level of pro-neurotensin orfragments thereof or pro-neurotensin 1-117 comprising peptide having atleast a length of 5 amino acids is determined by a diagnostic assay,preferably by an immunoassay.

In a specific embodiment of the method according to the invention themethod is performed more than once in order to monitor the risk ofgetting new-onset obesity or in order to monitor the course of treatmentof said subject to reduce the risk of getting new-onset obesity in asubject wherein said subject is non-obese.

In a specific embodiment of the method according to the invention saidmonitoring is performed in order to evaluate the response of saidsubject to preventive and/or therapeutic measures taken.

In a specific embodiment of the method according to the invention themethod is used in order to stratify said subjects into risk groups.

Also encompassed by the present invention is a point-of-care device forperforming a method according to the invention.

Also encompassed by the present invention is an assay and/or kit forperforming a method according to the invention.

EXAMPLES Example 1

Development of Antibodies

Peptides/Conjugates for Immunization:

Peptides for immunization were synthesized (JPT Technologies, Berlin,Germany) with an additional N-terminal Cysteine residue for conjugationof the peptides to Bovine Serum Albumin (BSA). The peptides werecovalently linked to BSA by using Sulfo-SMCC (Perbio-Science, Bonn,Germany) The coupling procedure was performed according to the manual ofPerbio.

Labelled antibody (LA) peptide (P-NT 1-19): H-CSDSEEEMKALEADFLTNMH-NH2Solid phase antibody (SPA) peptide (P-NT 44-62):H-CNLNSPAEETGEVHEEELVA-NH2

The antibodies were generated according to the following method:

A BALB/c mouse were immunized with 100 μg Peptide-BSA-Conjugate at day 0and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μg atday 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three daysbefore the fusion experiment was performed, the animal received 50 μg ofthe conjugate dissolved in 100 μl saline, given as one intraperitonealand one intravenous injection.

Splenocytes from the immunized mouse and cells of the myeloma cell lineSP2/0 were fused with 1 ml 50% polyethylene glycol for 30s at 37° C.After washing, the cells were seeded in 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium (RPMI 1640 culturemedium supplemented with 20% fetal calf serum and HAT-Supplement). Aftertwo weeks the HAT medium is replaced with HT Medium for three passagesfollowed by returning to the normal cell culture medium.

The cell culture supernatants were primary screened for antigen specificIgG antibodies three weeks after fusion. The positive testedmicrocultures were transferred into 24-well plates for propagation.After retesting the selected cultures were cloned and recloned using thelimiting-dilution technique and the isotypes were determined. (Lane, R.D. “A short-duration polyethylene glycol fusiontechnique for increasingproduction of monoclonal antibody-secreting hybridomas”, J. Immunol.Meth. 81: 223-228; (1985), Ziegler, B. et al. “Glutamate decarboxylase(GAD) is not detectable on the surface of rat islet cells examined bycytofluorometry and complement-dependent antibody-mediated cytotoxicityof monoclonal GAD antibodies”, Horm. Metab. Res. 28: 11-15, (1996)).

Monoclonal Antibody Production

Antibodies were produced via standard antibody production methods (Marxet al, Monoclonal Antibody Production, ATLA 25, 121, 1997) and purifiedvia Protein A-chromatography. The antibody purities were >95% based onSDS gel electrophoresis analysis.

Example 2

Immunoassay for the Quantification of Human Pro-Neurotensin

The technology used was a sandwich coated tube luminescence immunoassay,based on Akridinium ester labelling.

Labelled compound (tracer): 100 μg (100 μl) LA (1 mg/ml in PBS, pH 7.4,was mixed with 10 μl Akridinium NHS-ester (1 mg/ml in acetonitrile,InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at roomtemperature. Labelled LA was purified by Gel-filtration HPLC on Bio-SilSEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified LA was dilutedin (300 mmol/l potassiumphosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5g/l Bovine Serum Albumin, pH 7.0). The final concentration was approx.800.000 relative light units (RLU) of labelled compound (approx. 20 nglabeled antibody) per 200 Acridiniumester chemiluminescence was measuredby using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

Solid phase: Polystyrene tubes (Greiner Bio-One International AG,Austria) were coated (18 h at room temperature) with SPA (1.5 μg SPA/0.3ml 100 mmol/l NaCl, 50 mmol/l TRIS/HCl, pH 7.8). After blocking with 5%bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuumdried.

Calibration:

The assay was calibrated, using dilutions of pro-NT-containing humanserum. A pool of human sera with high pro-NT-immunoreactivity (InVentDiagostika, Hennigsdorf, Germany) was diluted with horse serum (BiochromAG, Deutschland) (assay standards).

The standards were calibrated by use of the human pro-NT-calibrator(ICI-Diagnostics, Berlin, Germany). Alternatively, the assay may becalibrated by synthetic or recombinant pro-NT 1-117 or fragments thereof(see also Ernst et al., 2006).

Pro-NT Immunoassay:

50 μl of sample (or calibrator) was pipetted into SPA coated tubes,after adding labelled LA (200 μl), the tubes were incubated for 16-22 hat 18-25° C. Unbound tracer was removed by washing 5 times (each 1 ml)with washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

Tube-bound LA was measured by using the AutoLumat LB 953.

FIG. 1 shows a typical P-NT dose/signal curve.

Example 3

Population Study

The Malmö Diet and Cancer (MDC) study is a population-based, prospectiveepidemiologic cohort of 28,449 men (born 1923-1945) and women (born1923-1950) from the city of Malmö in southern Sweden who underwentbaseline examinations between 1991 and 1996 (Minisymposium: The MalmoDiet and Cancer Study. Design, biological bank and biomarker programme JIntern Med 233, 39-79 (1993). From this cohort, 6,103 persons wererandomly selected to participate in the MDC Cardiovascular Cohort(MDC-CC), which was designed to investigate the epidemiology of carotidartery disease, between 1991 and 1994 (Persson et al. 2008.Atherosclerosis 200: 191-198). Fasted plasma samples at the baselineexamination were available for analysis of pro-neurotensin (pro-NT) andsuccessfully measured in a total of 4,632 participants in the MDC-CC. Ofthose, complete data was available for BMI in 4,626, for waistcircumference on 4,625 and for estimated degree of insulin resistanceusing the homeostasis model assessment of insulin resistance (HOMA-IR)(fasting blood glucose concentration×fasting plasma insulinconcentration/22.5) (Matthews et al. 1985. Diabetologia 28: 412-419) in4,468 participants. BMI was defined as body weight in kilograms dividedby the square of height in meters and obesity as a BMI ≥30 kg/m².Abdominal obesity was defined as a waist circumference of ≥94 cm inmales and ≥80 cm in females, according to the International DiabetesFederation definition (Alberti et al. 2006. Diabet Med 23: 469-489).Insulin resistance was regarded present in subjects belonging to the top25% of HOMA-IR values in the MDC-CC. ‘New-Onset Obesity’ is defined asobesity development among non-obese MDC-CC participants who werere-examined and diagnosed with obesity after an average follow-up timeof 16.5±1.5 years.

Pro-NT was measured in stored fasting plasma specimens that were frozento −80° C. immediately at the MDC-CC baseline exam using a recentchemiluminometric sandwich immunoassay to detect a pro-NT precursorfragment (pro-NT 1-117) as described previously (Ernst et al. 2006.Peptides 27: 1787-1793). Analyses of blood glucose and plasma insulinwere carried out at the time of baseline examination at the Departmentof Clinical Chemistry, Malmö University Hospital, which is attached to anational standardization and quality control system (Enhorning et al.2010. Circulation 121: 2102-2108). Of the 4,626 subjects with baselinedata on BMI and pro-NT, 2,900 subjects were re-examined with a newmeasurement of BMI after a mean follow-up of 16.5±1.5 years. In analysesof incident obesity, we excluded subjects who were obese already at thebaseline examination, leaving a total of 2,606 non-obese subjects foranalysis of pro-NT in relation to incident obesity. All participantsgave written informed consent and the study was approved by the EthicalCommittee at Lund University, Lund, Sweden.

Statistical Analyses

All subjects at the MDC-CC baseline examination were divided intoascending quartiles according to their value of fasting pro-NT. In crosssectional analyses we related baseline quartile of pro-NT to thedichotomous outcome of obesity, abdominal obesity and insulin resistanceusing age and sex adjusted logistic regression models. In the analysesof incident obesity, we related baseline quartile of pro-NT to thedichotomous outcome of incident obesity using logistic regressionadjusted for baseline age, sex and BMI. Data are presented as oddsratios (95% confidence intervals) and subjects belonging to the lowestquartile of pro-NT were defined as the referent group (odds ratio=1). ‘Pfor trend’ denotes the P-value for linear trend over quartiles 1-4.

Study Results

We measured pro-NT levels in fasted plasma of 4,632 middle-aged subjectsof the population-based Malmö Diet and Cancer Study CardiovascularCohort (Table 1). The age- and sex-adjusted likelihood of being obese,abdominally obese and insulin resistant significantly increased acrossquartiles of pro-NT (Table 2). Among non-obese subjects, the risk ofdeveloping obesity during an average follow-up time of 16.5±1.5 yearsincreased gradually with pro-NT quartiles, independently of baselinebody mass index, age and gender. Pro-NT median concentrations were 60.1pmol/L (range 3.3-75.9 pmol/L) in quartile 1, 89.3 pmol/L (range75.9-105 pmol/L) in quartile 2, 123 pmol/L (range 105-149 pmol/L) inquartile 3 and 190 pmol/L (range 149-1155 pmol/L) in quartile 4.Non-obese subjects in the top quartile of baseline pro-NT levels had amore than doubling (OR 2.06 (95% confidence interval of 1.38-3.06) ofthe risk of developing obesity as compared to subjects in the lowestquartile (Table 2).

Using the same variables in the equation, we investigated differentsubgroups for prediction of new-onset obesity (Table 3), subjects withdiabetes mellitus (DM) and impaired fasting glucose (IFG), high bloodpressure/anti-hypertensive therapy (AHT), metabolic syndrome (MeSy),eGFR <60 (ml/min/1.73 m²), heredity of cancer, prevalent cancer,smokers, respectively, at baseline were excluded. Non-obese subjects inthe highest pro-NT level quartile either none of the above mentionedconditions again showed a more than doubling of the risk of developingobesity compared to subjects in the lowest pro-NT quartile (Table 3).Non-obese subjects with none of these conditions (super healthysubjects) in the highest pro-NT level quartile even showed a more than3fold increased risk of developing obesity compared to subjects in thelowest pro-NT quartile.

Example 4

PNT Concentrations Before and after an Oral Fat Up-Take Test(“Cream-Test”)

A total of 54 patients, 19 healthy control subjects and 35 patients witha diagnosis of heart failure, were selected. The subjects fasted for atleast 10 hours and ingested a standardized fat-enriched drinkingsolution (cream containing 30% of fat). Blood was taken at baseline and1, 2 and 3 hours after cream up-take. Pro-Neurotensin was measured withthe immunoassay as described above. Baseline pro-NT was defined as 100%and levels at the three different time points were related thereto.Pro-NT significantly increased 1 hour after cream intake in both,healthy controls and patients with HF and decreased after 2 and 3 hoursbut without reaching the baseline level (FIG. 2). Moreover, the relativeconcentration of pro-NT was significantly different between healthycontrols and patients with HF at all three time points (p<0.05).

TABLE 1 Clinical characteristics of the Malmö Diet and CancerCardiovascular Cohort (MDC-CC) Characteristic Value N Age (years) 57.7 ±6.0  4,626 Female sex, n (%) 2661 (57.5) 4,626 Body Mass Index (kg/m²)25.8 ± 3.9  4,626 Waist circumference (cm) 84.0 ± 12.9 4,625 Fastingblood glucose (mM) 5.2 ± 1.4 4,468 Fasting insulin concentration (mU/L)7.0 (4.0-9.0) 4,468 HOMA-IR 1.5 (0.9-2.2) 4,468

Data are given as mean±standard deviation for normally distributedvariables, and as median and interquartile range for fasting insulinconcentration. Categorical data are presented as numbers (percentages).“N” denotes the number with complete data; thus, included in analyses.“HOMA-IR” stands for Homeostasis Model Assessment of Insulin Resistance(fasting plasma insulin concentration×fasting blood glucoseconcentration/22.5)

TABLE 2 Fasting plasma concentration of pro-neurotensin (pro-NT) inrelation to prevalence of obesity and insulin resistance and in relationto incidence of new-onset obesity during long term follow-up in theMalmö Diet and Cancer Cardiovascular Cohort Odds ratio (95% confidenceinterval) N/N Pro-NT Pro-NT Pro-NT Pro-NT P for cases Quartile 1Quartile 2 Quartile 3 Quartile 4 trend Prevalent 4626/604  1.0 1.00 1.131.34 0.01 obesity (ref) (0.78-1.29) (0.88-1.45) (1.05-1.70) Prevalent4625/1769 1.0 1.07 1.23 1.30 0.001 abdominal (ref) (0.90-1.27)(1.04-1.46) (1.09-1.54) obesity Prevalent 4468/1140 1.0 1.30 1.43 1.70<0.0001 insulin (ref) (1.06-1.59) (1.17-1.74) (1.39-2.06) resistanceNew-onset 2606/335  1.0 1.44 1.83 2.06 <0.01 obesity (ref) (0.95-2.10)(1.21-2.65) (1.38-3.06)

‘N/N cases’ denotes ‘total number of subjects in the analysis/number ofcases with the disease in question.’ ‘Pro-NT’ denotes fasting plasmaconcentration of pro-neurotensin at the MDC-CC baseline examination.‘Pro-NT Quartiles 1-4’ defines the MDC-CC population quartiles (lowestto highest) of pro-NT. Data are presented as odds ratios (95% confidenceintervals) and subjects belonging to the lowest quartile of pro-NT weredefined as the referent group (odds ratio=1). ‘P for trend’ denotes theP-value for linear trend over quartiles 1-4. Prevalent means that thesubjects already had the ‘disease in question’ at baseline, whereas thesubjects with prevalent obesity where excluded in the analysis forrisk-prediction of new onset obesity.

TABLE 3 Fasting plasma concentration of pro-neurotensin (pro-NT) inrelation to incidence of new-onset obesity in different subgroups ofpatients during long term follow-up in the Malmö Diet and CancerCardiovascular Cohort Odds ratio (95% confidence interval) N/N Pro-NTPro-NT Pro-NT Pro-NT P for cases Quartile 1 Quartile 2 Quartile 3Quartile 4 trend all 2606/335 1.0 1.44 1.83 2.06 <0.01 (ref) (0.95-2.10)(1.21-2.65) (1.38-3.06) Male 1080/137 1.0 1.43 1.36 2.36 0.044 (ref)(0.79-2.59) (0.75-2.48) (1.29-4.30) Female 1526/198 1.0 1.45 2.23 1.880.019 (ref) (0.85-2.46) (1.32-3.75) (1.09-3.21) No hereditary 1456/1971.0 1.65 1.68 2.37 0.014 cancer (ref) (0.99-2.77) (0.99-2.85)(1.41-3.97) No prevalent 2362/301 1.0 1.36 1.96 2.1 0.001 cancer (ref)(0.89-2.06) 1.29-2.95) (1.38-3.19) BP <140 1631/197 1.0 1.29 1.88 2.030.023 (ref) (0.77-2.18) (1.13-3.15) (1.21-3.39) No AHT 2269/273 1.0 1.632.03 2.12 0.004 (ref) (1.05-2.53) (1.31-3.13) (1.35-3.35) BP <140/no1526/175 1.0 1.44 1.99 2.16 0.026 AHT (ref) (0.83-2.49) (1.15-3.42)(1.24-3.73) Non-smoker 2001/241 1.0 1.47 1.75 2.26 0.006 (ref)(0.93-2.32) (1.12-2.74) (1.42-3.61) No prevalent 2544/326 1.0 1.51 1.952.17 0.001 cardiac disease (ref) (1.00-2.26) (1.30-2.90) (1.44-3.26)eGFR >60 2319/320 1.0 1.36 1.77 2.22 0.001 (ref) (0.90-2.04) (1.18-2.64)(1.47-3.35) No IFG or DM 2152/254 1.0 1.44 2.03 2.46 <0.001 (ref)(0.91-2.28) 1.30-3.19) (1.55-3.90) No MeSy 2426/302 1.0 1.44 1.92 2.030.003 (ref) (0.96-2.18) (1.28-2.89) (1.34-3.07) Healthy all 1155/123 1.01.84 2.68 3.17 0.005 (ref) (0.95-3.57) (1.41-5.09) (1.57-6.37) HealthyFemale 681/74 1.0 1.35 2.96 3.46 0.015 (ref) (0.56-3.25) (1.28-6.86)(1.37-8.73)

‘N/N cases’ denotes ‘total number of subjects in the analysis/number ofcases with the disease in question.’ ‘Pro-NT’ denotes fasting plasmaconcentration of pro-neurotensin at the MDC-CC baseline examination.‘Pro-NT Quartiles 1-4’ defines the MDC-CC population quartiles (lowestto highest) of pro-NT. Data are presented as odds ratios (95% confidenceintervals) and subjects belonging to the lowest quartile of pro-NT weredefined as the referent group (odds ratio=1). ‘P for trend’ denotes theP-value for linear trend over quartiles 1-4.

Heredity of cancer means no known cancer in family history at baseline,no prevalent cancer means no diagnosis of cancer at baseline, noprevalent cardiac disease means no myocardial infarction, ischemic heartdisease, stroke, heart failure (acute or chronic heart failure), atrialfibrillation and atrial flutter at baseline, BP=blood pressure,AHT=anti-hypertensive therapy, eGFR=estimated glomerular filtrationrate, IFG=impaired fasting glucose, DM=diabetes mellitus, MeSy=metabolicsyndrome.

TABLE 4 Pro-Neurotensin concentration at baseline (fasting) and 1, 2 and3 hours after cream intake in healthy control subjects and subjects withheart failure (HF) pro-NT [in %] baseline 1 h 2 h 3 h control 100 173.5147.0 133.0 HF 100 227.5 201.8 179.3

PNT values are given in % related to the baseline value, which wasdefined for both groups as 100%, respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a typical P-NT dose/signal curve

FIG. 2 shows PNT level before and after cream up-take in patients withheart failure and a control group

1-17. (canceled)
 18. A method for determining fat processing activityand/or for predicting risk of obesity in a subject comprising:determining by an immunoassay a level of pro-neurotensin 1-117 (SEQ IDNO: 5) or a level of peptides that comprise the amino acid sequence ofpro-neurotensin 1-117 (SEQ ID NO: 5) in a sample of bodily fluidobtained from said subject; and correlating said level ofpro-neurotensin 1-117 (SEQ ID NO: 5) or said level of peptides thatcomprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5)with fat processing activity and/or a risk of incidence of obesity insaid subject, wherein a level above a threshold level is indicative ofan enhanced fat processing activity and/or predictive for an enhancedrisk of obesity, wherein said subject is not obese when said sample ofbodily fluid is taken from said subject, wherein the subject does nothave metabolic syndrome, and wherein said immunoassay comprises (i)contacting said sample with one or more antibodies, at least one ofwhich is labelled, that specifically bind to an epitope withinpro-neurotensin 1-117 (SEQ ID NO: 5), to form a complex between theantibody and pro-neurotensin 1-117 (SEQ ID NO: 5) or peptides thatcomprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO:5), and (ii) quantitating the level of the thus-formed complex.
 19. Amethod according to claim 18, wherein said subject is a non-prediabeticsubject.
 20. A method according to claim 18, wherein a fasting level ofpro-neurotensin 1-117 (SEQ ID NO: 5) or of peptides that comprise theamino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) isdetermined.
 21. A method according to claim 18, wherein the level ofpro-neurotensin 1-117 (SEQ ID NO: 5) is determined.
 22. A methodaccording to claim 18, wherein the subject is non-diabetic with fastingwhole blood glucose of less than 6.1 mmol/l but more than 5.4 mmol/l.23. A method according to claim 18, wherein the subject does not havecancer.
 24. A method according to claim 18, wherein the subject has nohistory of diagnosis of an acute cardiovascular event when said sampleof bodily fluid is taken from said subject wherein said cardiovascularevent is selected from myocardial infarction, stroke, and acute heartfailure.
 25. A method according to claim 18, wherein said subject is asubject with fasting whole blood glucose of less than 5.4 mmol/l.
 26. Amethod according to claim 18, wherein the risk of obesity is independentof a risk for contracting diabetes mellitus and/or metabolic syndrome.27. A method according to claim 18, wherein additionally at least oneclinical parameter is determined in the subject selected from age,gender, systolic blood pressure, diastolic blood pressure,antihypertensive treatment (AHT), waist circumference, waist-hip-ratio,current smoker, diabetes heredity and previous cardiovascular disease(CVD).
 28. The method according to claim 18, wherein the sample isselected from a blood sample, a serum sample, a plasma sample, acerebrospinal fluid sample, a saliva sample and a urine sample or anextract of any of the aforementioned samples.
 29. A method according toclaim 18, wherein said method is performed more than once in order tomonitor the risk of incidence of obesity.
 30. A method according toclaim 29, wherein said monitoring is performed in order to evaluate aresponse of said subject to preventive and/or therapeutic measurestaken.
 31. A method according to claim 18, wherein said method isperformed in a plurality of subjects in order to stratify said subjectsinto risk groups.
 32. A method according to claim 18, wherein saidthreshold level is at least 60 pmol/L pro-neurotensin (pro-NT).
 33. Amethod according to claim 18, wherein said threshold level is at least90 pmol/L pro-neurotensin (pro-NT).
 34. A method according to claim 18,wherein said threshold level is at least 123 pmol/L pro-neurotensin(pro-NT).
 35. A method according to claim 18, wherein said thresholdlevel is at least 180 pmol/L pro-neurotensin (pro-NT).
 36. A methodaccording to claim 18, wherein peptides that comprise the amino acidsequence of pro-neurotensin 1-117 (SEQ ID NO: 5) are selected frompeptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 7.37. A method for determining fat processing activity and/or forpredicting risk of obesity in a subject comprising: determining by animmunoassay a level of pro-neurotensin 1-117 (SEQ ID NO: 5) or a levelof peptides that comprise the amino acid sequence of pro-neurotensin1-117 (SEQ ID NO: 5) in a non-fasting or fasting sample of bodily fluidobtained from said subject, administering fat to said subject,determining by an immunoassay a level of pro-neurotensin 1-117 (SEQ IDNO: 5) or a level of peptides that comprise the amino acid sequence ofpro-neurotensin 1-117 (SEQ ID NO: 5) in a sample of bodily fluidobtained from said subject after fat-uptake, calculating a differencebetween said levels after and before fat-uptake, and correlating saiddifference between said levels after and before fat-uptake ofpro-neurotensin 1-117 (SEQ ID NO: 5) or of said peptides that comprisethe amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5) with fatprocessing activity and/or a risk of incidence of obesity in saidsubject, wherein a higher difference is more indicative of an enhancedfat processing activity and/or more predictive of an enhanced risk ofobesity than a lower difference, wherein the subject is not obese whenthe samples of bodily fluid are taken from said subject, and whereinsaid immunoassay comprises (i) contacting said sample with one or moreantibodies, at least one of which is labelled, that specifically bind toan epitope within pro-neurotensin 1-117 (SEQ ID NO: 5), to form acomplex between the antibody and pro-neurotensin 1-117 (SEQ ID NO: 5) orpeptides that comprise the amino acid sequence of pro-neurotensin 1-117(SEQ ID NO: 5), and (ii) quantitating the level of the thus-formedcomplex.
 38. A method comprising: preparing a sample comprising acomplex of (a) an antibody and (b) pro-neurotensin 1-117 (SEQ ID NO: 5)or a peptide that comprise the amino acid sequence of pro-neurotensin1-117 (SEQ ID NO: 5) in a bodily fluid obtained from a subject who isnot obese when said sample of bodily fluid is taken from said subjectand said subject does not have metabolic syndrome.
 39. The methodaccording to claim 37, wherein the level of pro-neurotensin 1-117 (SEQID NO: 5) or a peptide that comprise the amino acid sequence ofpro-neurotensin 1-117 (SEQ ID NO: 5) in the bodily fluid of said subjectis above at least 60 pmol/L pro-neurotensin (pro-NT).
 40. A methodcomprising: preparing a first composition comprising a complex of (a) anantibody and (b) pro-neurotensin 1-117 (SEQ ID NO: 5) or a peptide thatcomprise the amino acid sequence of pro-neurotensin 1-117 (SEQ ID NO: 5)in a non-fasting or fasting sample of bodily fluid obtained from asubject who is not obese when said sample of bodily fluid is taken fromsaid subject; administering fat to said subject; and after administeringfat to said subject, preparing a second composition comprising a complexof (a) an antibody and (b) pro-neurotensin 1-117 (SEQ ID NO: 5) or apeptide that comprise the amino acid sequence of pro-neurotensin 1-117(SEQ ID NO: 5) in a sample of bodily fluid obtained from said subject.41. The method according to claim 40, wherein said first compositioncomprises a non-fasting sample of said bodily fluid.
 42. The methodaccording to claim 40, wherein said first composition comprises afasting sample of said bodily fluid.